CN112656760B

CN112656760B - Difluprednate suspension eye drops and preparation method thereof

Info

Publication number: CN112656760B
Application number: CN201910924146.8A
Authority: CN
Inventors: 牟东升; 周小顺; 刘文双; 吴欢; 魏志维; 蔡翠
Original assignee: Wuhan Conform Pharmaceutical Co ltd
Current assignee: Wuhan Conform Pharmaceutical Co ltd
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2022-04-12
Anticipated expiration: 2039-09-27
Also published as: CN112656760A

Abstract

The invention discloses difluprednate suspension eye drops and a preparation method thereof, wherein the difluprednate suspension eye drops comprise, by mass, 0.05-0.15% of difluprednate, 1-2.5% of modified collagen, 0.3-0.8% of medium-chain fatty glyceride, 0.2-0.8% of caprylic/capric polyethylene glycol glyceride, 0.2-0.6% of polyoxyethylene hydrogenated castor oil, 2.2% of glycerol, 0.1% of benzalkonium chloride and the balance of deionized water; the modified collagen is prepared by putting collagen powder into a closed stirring container, introducing mixed steam of chlorine dioxide and nitrogen, continuously stirring, and synchronously performing ultraviolet irradiation. The invention utilizes the adhesiveness of the modified collagen to obviously prolong the detention time of the drug in the eyes, can reduce the administration frequency and improve the drug compliance of patients with eye surgery compared with the common eye drops, and simultaneously ensures that the eyes are always in a drug microenvironment with higher concentration, thereby enhancing the treatment effect on the eye infection symptoms.

Description

Difluprednate suspension eye drops and preparation method thereof

Technical Field

The invention belongs to the field of ophthalmic preparations, and particularly relates to difluprednate suspension eye drops and a preparation method thereof.

Background

Difluprednate is a difluorinated derivative of the corticosteroid prednisolone, has a strong anti-inflammatory and analgesic effect, and is authorized to be an ophthalmic emulsion by Sirion corporation from kyoto pharmaceutical co. The clinical test of the difluprednate ophthalmic emulsion in the phase III obtains a positive result, and the difluprednate ophthalmic emulsion can quickly solve the ocular inflammation after the ophthalmic operation, and is safe and effective. The unique structure of the medicine enables the medicine components to rapidly enter the steroid horny layer, and can rapidly solve the problems of inflammation of anterior chamber cells and flashing of anterior chamber water.

Difluprednate is a fat-soluble drug, has poor solubility in water, and can be prepared into an ophthalmic emulsion to solve the problem of poor solubility. However, the retention time of the conventional emulsion on the cornea of the eye is short, and the long-acting property of the drug release is lacked. The common difluprednate ophthalmic emulsion on the market abroad needs to be dripped 4 times in one day initially when in use to ensure that the higher drug concentration of eyes is maintained, the drug administration frequency is poorer than the drug administration compliance of a patient who just undergoes eye surgery, the eyelid part can be pulled during drug administration, the healing of the wound of the eye surgery is not utilized in the frequent drug administration, the risk of wound infection is increased, and the workload of medical staff is increased by the multiple-frequency drug administration. An ophthalmic or otonasal composition containing difluprednate and levofloxacin provided in patent CN200910015868.8 and a glucocorticoid ophthalmic or otic-nasal suspension with redispersibility provided in patent CN201210259768.1, wherein glucocorticoid (including difluprednate, etc.) is mixed with conventional raw materials such as preservatives (boric acid, benzalkonium chloride, benzethonium chloride, sorbic acid, potassium sorbate, etc.), pH regulators (sodium acetate, phosphoric acid and salts thereof, etc.), surfactants (tween 80, carbomer, polyoxyethylene castor oil 60, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol stearate), various fatty oils (castor oil, etc.), glycerin, etc., according to the specification, the test method for simulating clinical surgical incisions of model rabbits requires continuous administration for 7 days, 4 times/day, and 2 drops/time; the frequency of administration is also high, by analogy to human patients. Therefore, the difluprednate ophthalmic preparation with sustained release, long-time effect and reduced administration frequency can better meet the clinical requirement.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides difluprednate suspension eye drops and a preparation method thereof, which can realize the purposes of slow release, long-time effect taking and administration frequency reduction, and are realized by the following technology.

The difluprednate suspension eye drops comprise, by mass, 0.05-0.15% of difluprednate, 1-2.5% of modified collagen, 0.3-0.8% of medium-chain fatty glyceride, 0.2-0.8% of caprylic/capric polyethylene glycol glyceride, 0.2-0.6% of polyoxyethylene hydrogenated castor oil, 2.2% of glycerol, 0.1% of benzalkonium chloride and the balance of deionized water;

the preparation method of the modified collagen comprises the following steps: the collagen powder is put into a closed stirring container, mixed steam of chlorine dioxide and nitrogen is introduced, the mixture is continuously stirred, and ultraviolet irradiation is synchronously carried out, so that the collagen powder is prepared.

The difluprednate in the difluprednate suspension eye drops consists of two parts: one part of the drug is dissolved in oil and surfactant to form nano emulsion, the rest part of drug is dispersed in the system in the form of nano particles under the action of emulsifier (caprylic/capric polyethylene glycol glyceride, polyoxyethylene hydrogenated castor oil), and stable and uniform suspension preparation is formed due to the stabilization effect of collagen. The difluprednate dissolved in the emulsion can be gradually absorbed by eyes in a short time to exert the drug effect; after the difluprednate suspended in the mixed solution is subjected to a high-pressure homogenization process, the difluprednate is solid nanoparticles and is easy to deposit on the cornea and inflammatory parts of eyes under the stabilizing and adhering actions of collagen, so that the difluprednate is gradually dissolved to release the medicine and is slowly absorbed by the eyes. The collagen is subjected to crosslinking modification under the oxygen-free condition by the synergistic effect of chlorine dioxide steam and ultraviolet rays, and compared with common collagen, the formed modified collagen can form a crosslinked net structure in a mixed solution to wrap and stabilize the difluprednate nanoemulsion and the difluprednate nanoparticles, has better adhesion, prolongs the detention time of the drug in eyes, prolongs the drug release time, ensures that the microenvironment of the eyes maintains higher drug concentration, can reduce the drug administration frequency, improves the drug administration compliance, and enhances the treatment effect.

Preferably, the difluprednate suspension eye drops comprise 0.1% of difluprednate, 1.8% of modified collagen, 0.6% of medium-chain fatty glyceride, 0.6% of caprylic/capric macrogol glyceride, 0.5% of polyoxyethylene hydrogenated castor oil, 2.2% of glycerol, 0.1% of benzalkonium chloride and the balance of deionized water in percentage by mass.

Preferably, in the preparation method of the modified collagen, the stirring time is 10-15 min, the stirring temperature is 35-45 ℃, and the stirring frequency is 100-150 rpm.

The invention also provides a preparation method of the difluprednate suspension eye drops, which comprises the following steps:

s1, stirring difluprednate, medium-chain fatty glyceride, polyoxyethylene hydrogenated castor oil and caprylic capric polyethylene glycol glyceride at 50-65 ℃ for 10-30min under a nitrogen protection environment to obtain an oil phase;

s2, under the nitrogen protection environment, stirring 50% of deionized water, benzalkonium chloride and glycerol for 5-10 min at 50-65 ℃ to obtain a water phase;

s3, slowly adding the oil phase into the water phase under the nitrogen protection environment, shearing at 5000-12000 rpm for 10-30min, and then homogenizing at 50-65 ℃ and 1000-1500 bar for 6-9 times to obtain a mixed solution;

s4, under the nitrogen protection environment, adding the rest 50% of deionized water into the modified collagen, stirring at normal temperature and dissolving;

and S5, under the nitrogen protection environment, pouring the modified collagen solution obtained in the step S4 into the mixed solution obtained in the step S3, and stirring at 40-50 ℃ and 300-800 rpm for 30min to obtain a finished suspension eye drop product.

According to the difluprednate suspension eye drops prepared by the invention, a part of difluprednate is dissolved in the emulsion, and undissolved difluprednate suspended in the system is easy to deposit on the cornea and inflammation parts of eyes under the action of stabilization and adhesion of collagen due to the fact that the difluprednate is high-pressure homogenized solid nanoparticles, so that the difluprednate can be gradually dissolved and released to be slowly absorbed by the eyes, a slow-release effect is achieved, meanwhile, the fluctuation of the drug concentration is reduced, and the microenvironment of the eyes keeps higher drug concentration for a long time.

Preferably, in step S3, the homogenization conditions are: homogenizing at 55 deg.C and 1300bar for 8 times.

Preferably, in step S5, the stirring condition is stirring at 45 ℃ and 600rpm for 30 min.

Compared with the prior art, the invention has the advantages that:

the difluprednate suspension eye drops prepared by the invention contain undissolved difluprednate nano-crystalline particles, the retention time of the drug in the eye is obviously prolonged by utilizing the adhesiveness of modified collagen and the stability of the modified collagen to the nano-crystalline particles, and meanwhile, the drug nano-crystalline particles can release the drug for a long time, so that the eye is always in a drug microenvironment with higher concentration, the drug frequency of eye surgery patients can be reduced, the drug compliance is improved, the times of pulling the vicinity of a surgical wound part due to drug administration are reduced, the wound healing is facilitated, the infection chance is reduced, and the curative effect is enhanced.

Drawings

FIG. 1 is a graph showing the in vitro cumulative release rates of the samples of examples 1 to 4 and comparative examples 2 and 4.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The modified collagen used in the following examples and comparative examples was prepared by the following method: putting collagen powder into a closed reaction kettle, introducing mixed steam of chlorine dioxide and nitrogen, continuously stirring, and synchronously performing ultraviolet irradiation to prepare the collagen powder; the stirring time is 10-15 min, the stirring temperature is 35-45 ℃, and the stirring frequency is 100-150 rpm.

Examples 1 to 3 and comparative examples 1 to 2

The raw materials of the difluprednate eye drops provided in the embodiments 1-3 and the comparative examples 1-2 are shown in the following table 1 in percentage by weight.

TABLE 1 composition table (%)

The difluprednate eye drops of the above examples 1-3 and comparative examples 1-2 are prepared by the following steps:

s1, stirring difluprednate, medium-chain fatty glyceride, polyoxyethylene hydrogenated castor oil and caprylic capric polyethylene glycol glyceride at 50-55 ℃ for 20min under the nitrogen protection environment to obtain an oil phase (with undissolved drug particles visible to naked eyes);

s2, stirring 50% of deionized water, benzalkonium chloride and glycerol at 55 ℃ for 8min under the nitrogen protection environment to obtain a water phase;

s3, slowly adding the oil phase into the water phase under nitrogen protection, shearing at 8000rpm for 20min, and homogenizing at 55 deg.C and 1300bar for 8 times to obtain mixed solution;

and S5, pouring the modified collagen solution obtained in the step S4 into the mixed solution under the nitrogen protection environment, and stirring at 45 ℃ and 600rpm for 30min to obtain the eye drop finished product.

In step S1 of comparative example 2, the drug was completely dissolved, and undissolved drug particles were not observed.

The difluprednate suspension eye drops of the comparative example 3 have the same components as those in the example 1, but have different preparation methods, and the difluprednate suspension eye drops are prepared by firstly adding the modified collagen and then homogenizing under high pressure, wherein the specific preparation method comprises the following steps:

s3, under the nitrogen protection environment, adding the rest 50% of deionized water into the modified collagen, stirring at normal temperature and dissolving;

s4, adding the collagen solution obtained in the step S3 into an S2 water phase under the nitrogen protection environment;

s5, slowly adding the oil phase into the water phase obtained in S4 under nitrogen protection, shearing at 8000rpm for 20min, and homogenizing at 55 deg.C and 1300bar for 8 times to obtain suspension eye drop.

Example 4

The kind and amount of raw materials of the difluprednate suspension eye drops provided in this example were exactly the same as those of example 1. The suspension eye drops were prepared in substantially the same manner as in example 1, except that in step S3, the homogenization conditions were as follows: homogenizing at 50 deg.C and 1500bar for 9 times.

Example 5

The kind and amount of raw materials of the difluprednate suspension eye drops provided in this example were exactly the same as those of example 1. The suspension eye drops were prepared in substantially the same manner as in example 1, except that in step S3, the homogenization conditions were as follows: homogenizing at 65 deg.C and 1000bar for 6 times.

Example 6

The kinds and the amounts of the components of the difluprednate suspension eye drops provided in this example are exactly the same as those of example 1. Suspension eye drops were prepared by substantially the same method as in example 1, except that in step S5, stirring was carried out at 40 ℃ and 800rpm for 30 min.

Comparative example 4

The difluprednate suspension eye drop provided by the comparative example has completely the same component types and dosage as those of example 1, except that the common collagen adopted by the comparative example comprises 0.1% of difluprednate, 1.8% of common collagen, 0.6% of medium-chain fatty glyceride, 0.6% of caprylic/capric polyethylene glycol glyceride, 0.5% of polyoxyethylene hydrogenated castor oil, 2.2% of glycerol, 0.1% of benzalkonium chloride and the balance of deionized water in percentage by mass.

In the method for preparing difluprednate suspension eye drops, the modified collagen of step S4 is replaced by normal collagen, and the other steps are the same as in example 1.

Comparative example 5

The difluprednate suspension eye drops provided by the comparative example have the same components as those in example 1, and the specific preparation method is basically the same as that in example 1, except that:

in step S3, the homogenization is carried out at 55 ℃ and 800bar for 5 times.

Application example 1: stability observation of Difluprednate eye drops of examples 1 to 6, comparative examples 1, 3 and 5

The eye drops prepared in examples 1 to 6 and comparative examples 1, 3 and 5 were placed in an environment of 40 ℃. + -. 2 ℃ and 75. + -. 5% relative humidity for 1 month, and the stability was evaluated by observing the appearance of the sample, and the test results are shown in Table 2 below.

TABLE 2 stability Observation of suspended eye drops

The data in Table 2 show that the difluprednate suspension eye drops prepared in examples 1-6 have good stability. The sample of comparative example 1 was unstable because it contained no collagen. The sample of comparative example 3, although containing collagen, was unstable, and the reason for the analysis was probably that the crosslinked structure of collagen was destroyed after high-pressure homogenization, resulting in a decrease in the stabilization thereof and thus causing instability of the sample. The sample of comparative example 5 was also unstable, probably because the homogenization pressure was insufficient, resulting in the drug particles being crushed insufficiently small, small amounts of large particles of the drug being difficult to observe in the system, and the Walsh ripening effect between these larger particles and the small particles in the system regenerated aggregates of large particles, so that large, visibly apparent drug particles appeared in the system after a period of standing.

Application example 2: in vitro Release test for eye drops of examples 1 to 4 and comparative examples 2 and 4

The appearance of the test sample was observed before the start of the test, and the particle size was measured, the results of which are shown in Table 3.

TABLE 3 test sample appearance and average particle size (nm)

Sample numbering	Appearance of the sample	Average particle diameter (nm)
			Example 1	The sample is uniform milk white, has no delamination, and has no visible drug particles	212.5
Example 2	The sample is uniform milk white, has no delamination, and has no visible drug particles	195.8
			Example 3	The sample is uniform milk white, has no delamination, and has no visible drug particles	227.6
Example 4	The sample is uniform milk white, has no delamination, and has no visible drug particles	201.3
			Comparative example 2	The sample is uniform milk white, has no delamination, and has no visible drug particles	192.9
Comparative example 4	The sample is uniform milk white, has no delamination, and has no visible drug particles	205.1

The data in Table 3 above show that the samples 1 to 4 of examples and the samples of comparative examples 2 and 4 have no difference in appearance and are consistent in average particle size.

The medicine release test is performed by using a diffusion cell, and using 5 μm artificial fiber membrane as permeation material, 20% ethanol solution as receiving medium, the dosage of the medicine supplied to the chamber is 0.2mL, the receiving cell volume is 7mL, and the effective permeation area is 3.14cm². The diffusion cell is placed in a constant temperature water bath (37 +/-0.5 ℃), and a magnetic stirrer is placed under the constant temperature water bath at the stirring speed of 300 r/min. 5mL of the receiving solution was taken out at 2, 4, 6, and 8 hours, respectively, and the same volume of the blank receiving solution was added. The obtained sample was centrifuged, and the supernatant was collected and analyzed by liquid chromatography to plot the cumulative release rate of difluprednate, as shown in fig. 1.

As can be seen from FIG. 1, the samples 1 to 4 in the embodiment of the present invention all have a good sustained release effect. The drug in the sample of comparative example 2 is mostly released and does not have the function of sustained release and long-acting as compared with the sample of the present invention. Although the sample of comparative example 4 has a certain sustained-release effect compared with the sample of comparative example 2, the effect of the sustained-release effect is obviously inferior to that of the sample of the embodiment of the present invention. The data in fig. 1 show that the drug is not sustained after being completely dissolved (as in the data of comparative example 2 in fig. 1), and the sustained release effect is not ideal when the conventional collagen is used (as in the data of comparative example 4 in fig. 1).

The sustained release effect of the present invention is likely to be drug nanoparticles derived from modified collagen and systems in suspension, demonstrating a surprising sustained release effect.

Application example 3: corneal residence test in eye for samples of examples 1 to 3 and comparative example 2

The diffusion cell is used for performing drug eye cornea retention experiment, rabbit eye cornea is used as a penetration material, 20% ethanol solution is used as a receiving medium, the dosage of a supply chamber is 0.2mL, the volume of a receiving cell is 7mL, and the effective penetration area is 3.14cm². The diffusion cell is placed in a constant temperature water bath (37 +/-0.5 ℃), and a magnetic stirrer is placed under the constant temperature water bath at the stirring speed of 300 r/min. After 4 hours of testing, the experiment was stopped, the cornea was removed, the suspension remaining on the surface of the cornea was washed clean, the cornea was cut into pieces, the drug in the tissue was extracted with an organic reagent and analyzed by liquid chromatography, and the results of corneal retention data for difluprednate eye are shown in table 4 below.

TABLE 4 corneal residual amount in eyes of samples of examples 1 to 3 and comparative example 2

Example 1	38.3％
		Example 2	37.4％
Example 3	33.6％
		Comparative example 2	3.8％

The data in table 4 show that the residual amount of the sample in the example on the cornea of the eye is significantly improved compared with that in comparative example 2, and the sample in the example can better ensure the drug concentration in the microenvironment of the eye and the long-term release of the drug.

Application example 4: examples 1, 4, 5 and comparative example 2

1. Test animal

Male Japanese big-ear white rabbits weighing about 2Kg and having no eye disease were used and kept in an animal house at room temperature of 24. + -. 2 ℃ with a daily fixed feed and with free water.

The mode of adopting the cornea incision of eye simulates the operation wound. In order to ensure the comparison of the drug effects and eliminate the influence caused by the difference of the drug concentration, the samples of examples 1, 4 and 5 and the sample of comparative example 2, in which the drug concentration is the same and is 0.1%, were used for the test.

2. Test method

(1) 20 healthy rabbits free from eye irritation, corneal defect and conjunctival damage were selected 24 hours before the test and randomly divided into 4 groups of 5 rabbits each of examples 1, 4 and 5 and comparative example 2.

(2) Fixing the rabbit, after local anesthesia of eyes, opening eyelids by using a sterilized eye dilator, cutting a straight incision with the length of about 3-5 mm and a small amount of slight aqueous humor on the corneal limbus as a base, injecting a quantitative pseudomonas aeruginosa liquid into a conjunctiva layer on the side of an operative incision by using an injector, simulating the condition of eye infection, and starting administration 24 hours after an operation.

The dose amounts in the examples 1, 4, 5 and comparative example 2 groups were: 1 drop/time, 1 time daily, continuously until day 6 after operation.

The rabbit operation parts and conjunctiva are observed by using a handheld slit lamp 48h, 96h and 144h after the operation and the first administration of the above groups, and the observation indexes are as follows: whether the surgical site is inflamed or not and whether the cornea and the conjunctiva are edematous or not are judged according to the specific scoring standards shown in the following table 5.

TABLE 5 Observation indexes and specific scoring standards of rabbit surgical site and conjunctiva

Eye symptom reaction	Score value
		A. Redness and swelling (finger operation site, palpebral conjunctiva and bulbar conjunctiva site)	——
Normal blood vessel color	0
		Blood vessel congestion is red, incision is red, and slight eversion	1
Blood vessel congestion is red, blood vessels are not easily distinguished, and wound is everted or protruded	2
		Diffuse hyperemia with purplish red color and severe protrusion of wound eversion	3
B. Edema (edema)	——
		Without edema	0
Minor edema (including instant membrane)	1
		Marked edema with partial outward turning of the eyelids	2
Edema to near-semi-closure of eyelid	3

3. Results and conclusions

(1) Daily observation scoring results

From 24 hours after the operation, before the first administration, 48 hours, 96 hours and 144 hours after the first administration are observed by using a handheld slit lamp, and whether the operation part is inflamed, red and swollen or not and whether the edema exists or not are respectively scored according to the scoring standard of the table 5.

Red and swollen symptom: the redness and swelling of the surgical incision and the symptoms of the congestion in the eyes represent the postoperative infection degree, and the indexes are used for scoring the redness and swelling of the surgical incision and the eyelid and observing the healing degree of the incision. The scoring results are shown in tables 6 and 7 below.

TABLE 6 evaluation results of rabbit surgical site and conjunctival redness symptom scores in examples 1, 4, 5 and comparative example 2

	Example 1	Example 4	Example 5	Comparative example 2
					Before the first administration	3	3	3	3
2 days after the first administration	2.2	2.2	2	2.6
					2 days after the second administration	0.6	0.8	0.6	2.0
2 days after the third administration	0.2	0.4	0.2	1.6

TABLE 7 evaluation results of rabbit surgical site and conjunctival edema symptoms in examples 1, 4, 5 and comparative example 2

	Example 1	Example 4	Example 5	Comparative example 2
					Before the first administration	3	3	3	3
2 days after the first administration	2.2	2.2	2.4	2.8
					2 days after the second administration	1	1.2	0.8	1.8
2 days after the third administration	0	0	0	1.4

As can be seen from tables 6 and 7, the rabbits to which the eye drops of examples 1, 4 and 5 were added had significant effects on the treatment of redness and swelling and edema of the operative site and conjunctiva, and the duration of the drug effect was long, and even after 1 day of single application, the drug effect was very good. The rabbit to which the eye drops of comparative example 2 were added had a poor therapeutic effect on the symptoms of redness and edema, and the score was significantly inferior to that of the animals of examples 1, 4 and 5. In combination with the data of in vitro release of application example 2, it is presumed that the sample prepared according to the present invention, in which some undissolved drug particles were contained in the operation of step S1, is advantageous to prolong the release of the drug and ensure the drug concentration in the microenvironment of the eye, whereas difluprednate of comparative example 2, which is completely dissolved, is released more rapidly and thus affects the therapeutic effect.

Claims

1. The difluprednate suspension eye drops are characterized by comprising, by mass, 0.05-0.15% of difluprednate, 1-2.5% of modified collagen, 0.3-0.8% of medium-chain fatty glyceride, 0.2-0.8% of caprylic/capric polyethylene glycol glyceride, 0.2-0.6% of polyoxyethylene hydrogenated castor oil, 2.2% of glycerol, 0.1% of benzalkonium chloride and the balance of deionized water;

the preparation method of the modified collagen comprises the following steps: putting collagen powder into a closed stirring container, introducing mixed steam of chlorine dioxide and nitrogen, continuously stirring, and synchronously performing ultraviolet irradiation to prepare the collagen powder;

the preparation method of the difluprednate suspension eye drops comprises the following steps:

s3, slowly adding the oil phase into the water phase under the nitrogen protection environment, shearing at 5000-12000 rpm for 10-30min, and homogenizing at 50-65 ℃ and 1000-1500 bar for 6-9 times to obtain a mixed solution;

2. The difluprednate suspension eye drops as claimed in claim 1, which comprise 0.1% of difluprednate, 1.8% of modified collagen, 0.6% of medium-chain fatty glyceride, 0.6% of caprylic/capric polyethylene glycol glyceride, 0.5% of polyoxyethylene hydrogenated castor oil, 2.2% of glycerol, 0.1% of benzalkonium chloride and the balance of deionized water in percentage by mass.

3. The difluprednate suspension eye drops as claimed in claim 1 or 2, wherein in the preparation method of the modified collagen, the stirring time is 10-15 min, the stirring temperature is 35-45 ℃, and the stirring frequency is 100-150 rpm.

4. The difluprednate suspension eye drops of claim 1, wherein in step S3, the homogenization conditions are as follows: homogenizing at 55 deg.C and 1300bar for 8 times.

5. The difluprednate suspension eye drops according to claim 1, wherein the stirring condition in step S5 is stirring at 45 ℃ and 600rpm for 30 min.